Drugs targeting the estrogen receptor (ER) in breast cancer have been extremely successful in controlling the disease for many patients, however acquired resistance is frequently encountered. We have identified recurrent mutations in the ligand binding domain (LBD) of ER among a high percentage of patients with such acquired resistance. We have found that the two most frequent mutations promote an agonist conformation despite the absence of ligand, cause resistance to aromatase inhibitors (AI), associate with poor clinical outcomes, and may be targeted by certain ER antagonists. However, we have more recently found that a spectrum of clinical ESR1 mutations exists. Our data reveal that there is diversity in the mechanisms whereby different mutants alter ER function as well as diversity in the impact of different mutations on ER conformation and activity. The overall hypothesis of this project is that different ER LBD mutations share an ability to promote some level of estrogen-independent receptor activity but have distinctive potencies in driving tumor phenotypes and promoting drug resistance. In this proposal, we will establish the mechanisms whereby different ESR1 mutations promote ER activity, characterize the gene expression programs driven by different mutations, understand the implications of different mutations for sensitivity to ER inhibition, identify likely routes of resistance to ER antagonists in ER mutant disease, and develop rational combinations to durably treat ER mutant cancer. To accomplish these goals, we will generate cell line models into which ER mutants have been knocked-in and patient derived organoid and xenograft models. We will evaluate existing ER antagonists and develop novel inhibitors to identify compounds that potently inhibit various ER mutants. Finally, we will use in vitro screens and tumor biopsies to characterize likely mechanisms of resistance to newer ER antagonists and nominate pharmacologic strategies to overcome these.